Class II MHC (Ia) gene products play critical roles in a variety of T and B lymphocyte responses. Biochemical and functional analyses have been used to investigate the relationship between class II structure and peptide antigen presentation. Class II molecules were found to undergo a characteristic structural change upon binding of appropriate peptides. The kinetics of class II peptide binding have been re-examined and two distinct phases of binding identified; a rapid initial binding with a rapid off rate, and a slow accumulation of long-lived binary complexes which is markedly augmented at the pH of the class II (endosomal) processing compartment. Studies in invariant chain-deficient mice have shown the critical role of this non-polymorphic protein in regulating class II assembly and folding within cells, as well as the capture of peptides derived from exogenous antigens. These new insights into the biochemical behavior of class II provide new understanding of how antigen capture by class II molecules occurs within living cells. In addition to binding peptide and being recognized by the clonally distributed T cell receptors, class II molecules participate in T cell selection in the thymus and activation in the periphery by interacting with the CD4 molecule that is also the receptor for HIV-1. We have used site-directed mutagenesis to define the site(s) of interaction of class II molecules with CD4, and demonstrated that a short continuous region in the beta2 domain of the class II molecules plays a major role in this interaction. We have also tentatively mapped a second region affecting CD4-class II interaction to the class II alpha chain. This latter observation may relate to the discovery that class II molecules form dimers of dimers when crystallized, and provides a new paradigm for assembly of the minimal T cell receptor signaling complex. Together with the studies on class II folding, transport, and control of peptide binding by the polymorphic domain, these experiments will provide new insight into the molecular mechanisms involved in antigen recognition by, and activation of T lymphocytes.